High-throughput MicroRNAomes Analysis In Melopsittacus Undulatus Gonads

Micro RNAs play important roles in gene expression that regulate post-transcriptionally in miscellaneous kinds of animals,plants,and viruses.Each micro RNA can target hundreds of messenger RNAs(m RNAs)in diverse binding sites through up-and down-regulation.Animal micro RNA functions like developmental timing,neuronal cell fate,cell proliferation and differentiation,metabolism and ageing,apoptosis and organ morphogenesis which suggest that mi RNAs are especially significant for the control of animal birth and death.There are currently numerous micro RNA researches on plants and mammals,however,seldom on aves.In order to enrich the micro RNA infromation from aves,we isolated total RNAs of M.undulates from the ovary of female adult(n=3)and the testis of male adult(n=3)to construct two small RNA libraries,and sequenced them using Illumina Hiseq 2500 in a single lane.Based on the analysis of the total reads,we try to address:(1)The micro RNA profile on M.undulates,(2)The comparative analysis of the micro RNA dataset in M.undulates.(3)The screening of the differentially expressed micro RNA from ovary and testis tissues of M.undulates.(4)The evolutionary of micro RNA.The main findings are as follows:1.Small RNA Sequence ProfileAfter filtering for adaptors and junk contaminants,the total reads of the ovary are 9,191,836,and that of the testis are 6,383,927,respectively.There were 82.03%(ovary)and 84.43%(testis)clean reads in these two libraries,respectively.Finally,7,540,057 cleaned sequences of ovary representing 572,201 unique reads and 5,389,781 cleaned sequences of testis representing 2,370,340 unique reads were used for subsequent analysis.2.Identification of New micro RNAsIn this study,142 of 151 total predicted pre-mi RNAs were sequenced and they coded 92 families including 211 new known micro RNAs,which 13 of 211 mi RNAs coded conserved mi RNA only in aves or first time in aves(conserved in other species),and other micro RNAs both in aves and other species across the budgerigar samples.3.The function of micro RNA family in GGU motif and the necessity for the regulative factorsIn this study,we examined all mi RNAs that 77 mi RNAs displayed internal nucleotide change at the U position within a GGU motif which could probably motify mi RNA characteristic target to have deep influence on relevant cellular procedure and gene expression(Luo,Hafner et al.2012),GGU motifs occurred within the seed sequence,and the others are outside the seed sequence for 17 of 77 mi RNAs in our study.4.qPCR validation and functional analysis of the avian specific mi R-2954This study demonstrates that target genes of conserved mi RNAs.mi R2954 is an avian specific mi RNA which expressed at higher ranks in males than in females,both in chicken.We got the same result from bioinformatics prediction as well as q PCR validation.Putative target genes of mi R2954 mainly encoded a TLE4 transcription factor family protein,which might be associated with particular functions in the nervous system,including Ca MKIV,SCAMP1,and SMARCA2.Interestingly,in KEGG analysis,Mun-mi R-2954 related to development,environmental adaptation,nervous system,signaling molecules and interaction,and substance dependence.These results provide valuable information for the further research.5.Significant differences in micro RNA expression and phylogenetic analysis in gonadsIn this study,we found 17 of 252 significant differences in micro RNA expression,such as Mun-novel6-3p,Mun-novel31-5p,Mun-mi R-1626-5p,Mun-mi R-215-5p and Mun-mi R-2954a-3p.We selected Mun-mi R215-5p using q PCR validation and based on the precursor structure sequences which released in mi RBase v21.0,we found the evolutionary sense from the phylogenetic analysis in pre-mi RNA.We provided the data of 252 pre-mi RNA for further analysis.In conclusion,we reported the first origin data for the whole mi RNAome of budgerigar.We also identified 211 known mi RNAs and 41 novel mi RNAs which every mi RNA family has a sexual specificity in physiological and functional processes.Moreover,we validated some differentially expressed mi RNAs(Mun-mi R-18b-5p,Mun-mi R-2954a-3p,Mun-mi R-215-5p,Mun-novel10-5p,Mun-novel24-3p)between ovary and testis randomly.mi RNAs have emerged as powerful regulatory molecules,from our analysis,some mi RNAs can influence sex-specific responses to potential biological mechanisms and functions for further analysis.